We hypothesize that the development of peripherally-restricted, mixed-activity opioid receptor agonists will produce robust analgesia while exhibiting reduced side effects, including a lack of respiratory depression or reinforcing properties. In preliminary studies, the in vitro screening of novel bis-imidazolidines and fused heterocyclic lead compounds BIM-22 and FDC-14 identified mixed-opioid receptor agonist activity. After peripheral (i.p.) administration to mice, both compounds demonstrated antinociception equivalent to morphine in the 55oC warm-water tail withdrawal (tail-flick) assay, but FDC-14 was 25 times more potent than morphine in the acetic acid writhing test, with an antinociceptive potency ratio of 72.7, suggestive of peripherally- restricted activity. Confirming this, LC-MS/MS studies detected BIM-22 and FDC-14 in harvested mouse blood, but not brain, after i.v. administration. This proposal addresses two research areas of particular interest for this FOA: i) The development of new and innovative molecular probes for receptors and new strategies for innovative peptidomimetic design, and ii) The identification of structurally diverse, orally active, metabolically stable peripherally-restricted opioid agonists. We propose six interacting aims: In Aim 1, we propose the computationally guided synthesis of heterocyclic peptidomimetics: bis-imidazolidin-2-imines, piperazines, bis- piperazine, bis-imidazolone and fused heterocyclic libraries. In Aim 2, we will screen all compounds in competition radioligand binding assays to determine affinity for ?- (MOR), ?- (DOR), and ?- (KOR) opioid receptors, and in functional assays to identify dual ?/? or dual ?/ agonists. In Aim 3, ten selected compounds will be evaluated in vitro for pharmacokinetic properties and screened in vivo for antinociception using the mouse tail-flick assay. Lead compounds identified from this aim will guide 2nd generation SAR studies to enhance peripherally-selective activity. In Aim 4: we will perform full in vivo antinociceptive characterization of lead agonist compounds. The most stable, active 4 agonists not crossing the BBB in Aims 2+3 will be evaluated after i.p. administration with mouse tail-flick and acetic-acid writhing assays for efficacy, duration of action, and opioid receptor selectivity. In Aim 5: we will characterize the bioavailability in mice of the selected 4 agonists following oral administration and confirm their inability to penetrate the blood brain barrier. In Aim 6: The two most potent bioavailable novel agonists identified in Aims 1-5 will be examined for liabilities, specifically antinociceptive tolerance, respiratory and hyperlocomotor effects in the CLAMS physiological and behavioral assessment system, sedation and disruption of coordinated locomotor activity in the rotorod assay, and assessment for rewarding or aversive effects in the conditioned place preference (CPP) assay. Effects on GI transit will also be evaluated to assess effects on constipation. In summary, we expect to generate novel peripherally-restricted, mixed-activity peptidomimetic opioid receptor agonists as both probes and analgesics with reduced side effects, thereby significantly impacting analgesic development.